Electrophoresis is a well known process for separation of charged species which utilizes different mobilities of these species in an electric field. The mobilities depend on the electrophoresis medium, electric field strength and characteristics of ions themselves, including net surface charge, size and shape. Small species, like metal ions, as well as large species such as viruses have been separated by electrophoretic techniques, but electrophoresis is currently used mostly for separation of biological macromolecules, including proteins, nucleic acids and their derivatives. The process is usually carried out by forcing the molecules to migrate through an aqueous gel. The gels used in electrophoresis are composed of natural or synthetic polymers. Agarose is the most widely used natural material and polyacrylamide gels represent the most common synthetic matrix. The gels are run in essentially two types of electrophoretic units: vertical and horizontal ones. In horizontal units the contact between the electrodes and the gel may be established directly or by means of wicks. Alternatively, in submerged gel electrophoresis the gel is immersed in buffer which serves as a conductive medium between electrodes and the gel. This format is the simplest and is widely used for analysis of nucleic acids. Agarose gels are almost exclusively used for submerged gel electrophoresis of nucleic acids. A new synthetic matrix has been introduced for analysis of proteins and nucleic acids by Kozulic et al (U.S. patent application Ser. No. 328,123, Analytical Biochemistry 163 (1987) 506-512 and Analytical Biochemistry 170 (1988) 478-484). It is based on an acrylic monomer, N-acryloyl,tris(hydroxymethyl)aminomethane (NAT). The poly(NAT) gels were found to be more porous than polyacrylamide gels but less porous than agarose gels. Therefore they offered advantages for separation of large proteins and those nucleic acids whose size is out of the optimal separation range of agarose and polyacrylamide gels. In the cited references, the superior properties of the poly(NAT) gels for analysis of DNA were demonstrated after running the gels in a vertical format. However, it was found that in the standard submerged electrophoresis units the resolution of DNA in the poly(NAT) gels was never so good as in the vertical system. The major difference was observed in the lower half of the gel, where the bands became much more diffuse. Moreover, the DNA fragments in the middle lanes migrated further than the corresponding fragments in the outer lanes. This phenomenon is known as the smiling effect. Further, very often DNA bands were straight only in the middle but the edges were bent upwards. The occurrences described above were eliminated when the gels were run in an improved apparatus for submerged gel electrophoresis (Kozulic and Heimgartner, UK Patent Application 9024428.6). In that apparatus buffer cooling and recirculation control the heat produced during electrophoresis and prevent buffer ion depletion in the electrode compartment. In addition, the electric field is more homogenous than in the standard submerged electrophoresis units. The combination of these three improvements has resulted in better resolution of DNA fragments in poly(NAT) gels.
The poly(NAT) gels run in the improved apparatus were usually three millimeters thick. The sample wells were formed with combs 2.5 mm deep, 1.5 mm wide and 5.5 mm long. Thus volume of the sample well should be about 20 .mu.l, but in practice it is about 15 .mu.l because the wells distort slightly after removal of the comb. When the sample volume was from 2 to 5 .mu.l, the resolution of DNA fragments was excellent but it was rather poor, as revealed by a photograph of the gel, when the sample volume was 10 .mu.l or more. That remit was in accordance with many reports in prior art showing that a small sample volume was essential for optimal resolution. Therefore, the poor resolution in submerged poly(NAT) gels at higher sample volumes was initially regarded as normal. Ethidium bromide was used to stain DNA in poly(NAT) gels and the fluorescence of DNA-ethidium bromide complexes was visualized under a UV light. It was by casualty observed that by looking at the gel from above under an angle declined from the vertical, the viewed DNA bands in the gel were much sharper than they were pictured on the photograph taken by a camera positioned more or less vertically above the gel. On basis of this observation it was assumed that the diffuseness of bands seen on the photograph did not originate from a real diffuseness of bands in the gel, but rather from the vertical position of the camera and bending of the bands against the vertical axis. As a consequence of this observation the following object of an invention has crystallized out: If the bands in the gel could be made essentially vertical, then the resolution taken by the usually positioned camera would be qualitatively better and besides that independent of the sample volume. It is now the main goal of the following discussion to describe factors causing bending of bands in gels during electrophoresis and practical means to greatly diminish such bending.